The aim of the research program is to elucidate one mechanism of action of insulin and to identify all of the cellular components necessary for the hormonal response. Because it has recently been discovered that the insulin receptor has tyrosine-specific protein kinase activity, the hypothesis is that phophorylation of tyrosine residues on select target proteins is responsible for some of the cellular actions of insulin. The strategy of this proposal is first to identify the components of an insulin-sensitive system and then to examine the potential regulation of these components by the insulin receptor. The insulin-stimulated phosphorylation of ribosomal protein S6 in 3T3-L1 adipocytes will be analyzed because 1) an S6 protein kinase has been found whose activity is greater in insulin-treated cells and 2) insulin receptor partially purified by chromatography on wheat germ agglutinin-agarose can stimulate the phosphorylation of S6 by crude S6 kinase preparations in an insulin-dependent manner. We will examine whether or not the insulin receptor activates the S6 protein kinase in vitro and if it does so directly by phosphorylation of tyrosine residues on the enzyme. The first experiments will employ purified preparations of the insulin receptor and the S6 protein kinase. If activation of S6 phosphorylation cannot be elicited by mixing the two enzymes, additional components of the more crude preparations will be assayed for their ability to reconstitute activation in the cell-free system. The objective is to generate an insulin-sensitive system composed entirely of pure constituents, so that their interactions can be fully characterized. We will also determine the role of the S6 protein kinase in the insulin-stimulated phosphorylation of the ribosomal protein in intact 3T3-L1 cells. This will be done by peptide mapping of the phosphopeptides derived from tryptic digests of S6 labeled in intact cells and in vitro and by sequence analysis of these phosphorylation sites. Finally, we plan to investigate the importance of other protein kinases and phosphoprotein phosphatases in this insulin response.